This project aims at the development of a novel method for the quantitative, objective, and rapid analysis of the expression of the molecule Her-2/neu on breast tissue biopsies. Her-2/neu is a prognostic and predictive biomarker for breast cancer, and its level of expression is a basis for the determination of the optimal treatment modality in contemporary clinical practice. The technology platform proposed herein employs characterization-mode ultrasound, in conjunction with immunotargeted micro- and nanoparticles, for the amplification of the molecular signature. The centerpiece of the approach is a novel theoretical framework, which enables the identification of previously inaccessible information on the physical properties of the probed tissue, at different length scales. Preliminary studies have yielded encouraging results on both native and particle-modified tissue samples. The hypothesis underlying the proposal is that molecular information can be translated into mechanical properties at specified length scales by way of nanoparticle immunotargeting, and that information based on these properties can be detected and analyzed using the ultrasonic characterization system and software proposed herein. In the R21 phase, we propose to establish the feasibility of this approach, starting with the development and refinement of robust protocols for the derivatization of nanoparticles with antibody conjugates; the development of sample preparation methods that are most suited for quantitative interrogation by ultrasonic characterization; the completion of an ultrasonic characterization system for detecting the nanoparticle signals; and the finalization of the data analysis tool under the novel, multi-scale mechanical field theory. In keeping with the proof-of-principle nature of this phase, the milestones proposed for the transition into the R33 phase are the attainment of specificity and sensitivity figures that are no less than those that pertain to prevalent current pathological investigation techniques, i.e., in this context, IHC and FISH. Even at identical sensitivity and specificity, the proposed system is believed to be potentially advantageous over IHC and FISH in that it is very rapid, automated, and objective, has high potential for interobserver and interlaboratory agreement, and requires significantly reduced level of investment of a specialist's time. In the R33 phase, the objectives of the proposed program would be to optimize the system's performance and create an actual prototype for clinical testing in the context of breast malignancies.